Peptide drugs and drugs containing a peptidase labile bond are among the most promising medicinal agents of modern times, but their instability in the presence of proteolytic enzymes in the gastrointestinal tract and other mucosal tissues usually requires that they be administered parenterally. Although patients can be taught to inject parenterally, there has been a long felt need to develop a non-invasive method for self administration of peptide drugs.
Protease inhibitors and penetration enhancers are means often considered to circumvent the enzymatic and penetration barriers to peptide and protein absorption from mucosal routes of administration. Because of such barriers, the bioavailability of peptide and protein drugs from mucosal routes is poor.
Non-parenteral administration of peptide drugs in particular often results in very low bioavailability because of hydrolysis of the peptides by proteolytic enzymes. For leuprolide, this ranges from 0.05% following oral administration to 38% following vaginal administration; for insulin, the corresponding figures are 0.05% and 18%. Lee, Journal of Controlled Release, 13, 213 (1990).
Examples of proteolytic enzymes which inactivate proteolytically-labile therapeutic agents are pepsin, trypsin, chymotrypsin, elastase, and carboxypeptidase in the intestinal lumen, and the aminopeptidases located on the mucosal surfaces of the GI tract, nose, and vagina.
Transport of intact oligopeptides across adult mammalian jejunum has been demonstrated in vitro and in vivo as well as in combination with peptidase inhibitors. Friedman and Amidon, Pharmaceutical Research 8, no. 1, P. 93, (1991).
Fujii et al; U.S. Pat. No. 4,639,435 (1987) claims the use of 1-isopropyl-4-[4-(1,2,3,4-tetrahydronaphthoyloxy)benzoyl] piperazine methanesulfonate as an inhibitor of chymotrypsin to be co-dosed orally or rectally with a chymotrypsin-labile drug (kallikrein or calcitonin). The reference also discloses the use of benzoylpiperazine esters for this purpose. The reference does not describe the mechanism of these inhibitors.
Cho and Flynn; International Patent Application WO-90/03164 (1990) disclose the use of protease inhibitors in oral formulation but do not describe the nature of such inhibitors in detail; the only protease inhibitor which appears in the examples is aprotinin.
Kidron, et al; U.S. Pat. No. 4,579,730 (1986) disclose the use of protease inhibitors in oral formulation of insulin. Soybean flour is disclosed as a source of soybean trypsin inhibitor (Bowman-Birk trypsin/chymotrypsin inhibitor; molecular weight 8000 daltons).
Ziv, et al; Biochem. Pharmacol. 36, 1035-1039 (1987) disclose the use of the protease inhibitor aprotinin to enhance the oral absorption of proteins.
Losse, et al; East German Patent DD 252 539 A1 (1987) disclose the use of epsilon-aminocaproic acid and aprotinin as protease inhibitors in oral formulation of peptides.
Lee; J.; Controlled Release 13, 213-223 (1990) reviews the use of protease inhibitors in formulations of peptides for oral, nasal, buccal, rectal, vaginal, pulmonary, and ocular routes.
Certain small peptides containing up to four amino acids have been shown to enhance the bioavailability of peptide drugs.
Hussain, et al, Biochemical and Biophysical Research Communications, 133, no. 3, 923 (195) suggested that nasal administration of peptides may become an important route provided that peptidases in the nasal mucosa can be transiently inhibited via coadministration of pharmacologically inactive peptidase substrates.
Faraj, et al, Journal of Pharmaceutical Sciences 79, no. 8, 698 (1990) showed that in the presence of the small peptides, L-tyrosyl-L-tyrosine and tri-L-tyrosine methyl ester, the hydrolysis of leucine enkaphalin was reduced, suggesting that competitive inhibition of nasal peptidases was caused by these small peptides.
Hori et al; J. Pharm. Sci. 72, 435-439 (1983), disclose the use of various amino-protected peptides to protect insulin from degradation when injected subcutaneously. Numerous publications disclose enzymatic treatment of vegetable proteins. An early U.S. Patent No. by John R. Turner (U.S. Pat. No. 2,489,208) discloses a pepsin modified whipping agent component. An alkaline material such as sodium sulfite, sodium carbonate or sodium hydroxide is used to extract glycinin at a pH 6.4-6.8. The glycinin is then precipitated from the extract (e.g., pH 4.2-4.6) at its isoelectric pH in which sulfur dioxide may be utilized as the adjusting acid. The precipitated glycinin product is then modified with pepsin under temperature and pH conditions conducive to hydrolysis of protein. The glycinin is hydrolyzed with pepsin until its water-solubility is increased to 40-50%. Similarly, U.S. Pat. No. 2,502,482 by Sair et al. reports the enzymatic modification of glycinin with pepsin to produce an isolate wherein at least 60% by weight of the pepsin modified isolate is water-soluble at a pH 5.0.
Puski reports the enzymatic modifying of soy isolates (precipitated at pH 4.5) with Aspergillus oryzae in "Modification of Functional Properties of Soy Proteins by Proteolytic Enzyme Treatment" (Cereal Chem. 52, pages 655-665 (1975)).
Several publications also report using saline solutions to extract soy proteins. A publication by A. K. Smith et al. (Jr. American Chemical Society, Vol. 60, June 1938, pages 1316-1320) reports the extraction of soybean meal with pH 6.7 water alone yields more protein extract than an aqueous extraction in the presence of neutral salts.
U.S. Pat. No. 4,131,607 by Petit discloses a two-stage alkaline extraction. The extraction is initially conducted in the presence of sodium sulphite and magnesium salt at a pH 7.0-8.5 which is then increased to a pH 10.0-10.5 to complete the extraction. The protein extras are then precipitated or curded by adjusting the extract to a pH 4.5-5.5. A patent issued to Martinez et al. (U.S. Pat. No. 3,579,496) similarly discloses a multiple solvent extraction process.